ANALYSIS OF DIRECT FACTOR Xa INHIBITORS

ABSTRACT

The present invention relates to a method for detecting at least one direct factor Xa inhibitor in a sample other than citrate plasma, comprising the step of mixing a sample containing a factor Xa inhibitor with a composition containing factor Xa under conditions which allow the factor Xa to release a detectable substance from a chromogenic substrate.

CROSS-REFERENCE

This application is a section 371 of International application no. WO2012/069139, filed Nov. 7, 2011 which claims priority from GB Patentapplication no. 1019674.9, filed Nov. 22, 2010, which is incorporated byreference in its entirety.

FIELD

The present invention relates to a method for detecting at least onedirect factor Xa inhibitor in a sample other than citrate plasma,comprising the step of mixing a sample containing a factor Xa inhibitorwith a composition containing factor Xa under conditions which allow thefactor Xa to release a detectable substance from a chromogenicsubstrate.

BACKGROUND

Blood coagulation tests are conducted on plasma samples fromhumans/animals by making blood samples incoagulable with sodium citrate(volume ratio blood/anticoagulant 9:1). To conduct the determination ofindividual measurement values of the blood coagulation, blood iscentrifuged and the cells (in the sediment) are separated from theliquid blood components (plasma). To measure the blood coagulation,calcium chloride and an activator of the blood coagulation are added tothe plasma. In special methods for determining individual clottingfactors or exogenous inhibitors, photometric proof is used. In thesemethods, the colorant para-nitroaniline is released by an exogenouslyadded coagulation enzyme (e.g. factor Xa) from a chromogenic substrate(N-benzoyl-L-isoleucyl-L-glutamyl-L-glycyl-L-arginine-para-nitroanilinehydrochloride and others), the activity/concentration of factor Xainhibitors or substances inhibiting factor Xa being measured in aconcentration-dependent manner. The result is a linear or sigmoidaldecrease of the release of para-nitroaniline, measured at 405 nanometersin the photometer, depending on the concentration of the factor Xainhibitor (Harenberg J, Modified anti-factor Xa chromogenic substrateassay for heparin and low molecular weight heparins Arztl. Lab. 1987,33: 39-41).

For other tests in blood, such as liver enzymes, kidney parameters,electrolytes or cholesterol, blood is coagulated by the addition of anactivator (e.g. kaolin). Here, the coagulation proteins, antithrombinand fibrinogen are used. These factors are mixed with blood cells in ablood clot in the coagulum in a coagulation tube, into which blood iswithdrawn. Serum can be found in the supernatant, which does not containthese coagulation proteins. The measurement of these clinical-chemicalparameters is easier in serum. Concentrations of drugs are measured inserum as well, except for the coagulation drugs for clinical use.

Heparins, low-molecular-weight heparins, heparinoids, fondaparinux andother polysaccharides need cofactors in the blood (antithrombin, heparincofactor II) to activate their anticoagulative effect or to accelerateit by as much as 1000 times. These cofactors are present in plasma, sothat citrate plasma samples are used for analyzing the activity of theinhibitors of blood coagulation. Tests in serum are not possible withmethods for the clinical routine (Harenberg J, Neue Antikoagulantien.Zett Verlag, Steinen, 2007).

Other inhibitors of blood coagulation do not need cofactors in the bloodto become active. These are so-called direct coagulation inhibitors ofcoagulation enzymes. The most important ones for the time being are thegroup of direct factor Xa and thrombin inhibitors. Theiractivity/concentration is measured in citrate plasma with differentmethods/activators. Rivaroxaban is the first one of the oral directfactor Xa inhibitors to be clinically used. The relevant determinationmethods for detecting the concentration/activity of rivaroxaban arepublished. All analyses are made with plasma anticoagulated with citrate(Samama M M, Martinoli J, Leflem L, et al. Assessment of laboratoryassays to measure rivaroxababan—on oral, direct factor Xa inhibitor.Thromb Haemost 2010; 103: 815-825; Tripodi A, Guinet C, Samama M. TheInternationalized Normalized Ratio (INR) Calibrated for Rivaroxaban(INRivaroxaban) Normalizes Prothrombin Time Results for Patients Treatedwith this Drug. J Thromb Haemost 2010, online available; Harenberg J,Marx S, Kramer R et al. Reduction of variability between prothrombintime reagents of plasma samples containing rivaroxaban using the WHO/RBT90 thromboplastin reagent, submitted for publication).

So far, there have not been any detection methods in theclinical-chemical routine for inhibitors of blood coagulation fromserum. Serum has different advantages over plasma: In medicine, serumsamples are more often taken from patients than plasma samples. Theblood withdrawal is less susceptible to influences for serum samplesthan for plasma samples. In the case of a “bad” blood withdrawal, bloodcoagulation can be activated. Thereby, the results are influenced by theclotting factors. This is not possible for serum samples, since a bloodcoagulation is performed in the tube after the withdrawal.

Current solutions have the disadvantage of requiring a separate bloodwithdrawal for obtaining plasma samples for the analysis. Antithrombin,factor X/Xa and other coagulation proteins are contained in thepatients' plasma in different quantities. This influences the testresult. A blood withdrawal involves the risk of local side effects, suchas hematoma, or generalized side effects, such as inflammation of thevein or transmission of infection (e.g. hepatitis, HIV).

Thus, the problem underlying the present invention is to provide newmeans for an efficient detection of factor Xa inhibitors which overcomethe shortcomings of the protocols known in the art.

SUMMARY

The solution to the above technical problem is achieved by theembodiments characterized in the claims.

In particular, the present invention relates to a method for detectingat least one direct factor Xa inhibitor in a sample, comprising thesteps of:

(a) providing a sample containing at least one direct factor Xainhibitor;(b) providing a composition containing factor Xa;(c) providing a composition containing a chromogenic substrateconjugated to a detectable substance;(d) mixing the sample of step (a) with the composition of step (b) andthe composition of step (c) under conditions which allow the binding ofthe at least one direct factor Xa inhibitor to factor Xa and which allowthe factor Xa to release the detectable substance from the chromogenicsubstrate;(e) measuring the amount of released detectable substance,wherein the sample does not contain citrated blood plasma.

In other embodiments of the present invention, a method for monitoringthe course of treatment with factor Xa inhibitors, a composition, and adiagnostic kit are also provided.

DESCRIPTION OF THE DRAWINGS

The figures show:

FIG. 1: Overview of the reactants in the test system and the reactionprocess and the measurement in a preferred embodiment of the presentinvention.

FIG. 2: Illustration of the inhibition of factor Xa by increasingquantity of rivaroxaban (x axis) in plasma (method so far), in serum andurine (new methods). Small quantities of rivaroxaban inhibit little/nofactor Xa, so that much colorant is released from the chromogenicsubstrate (high absorption, y axis, nm=nanometer of the wavelength formeasuring the colorant). The less rivaroxaban is needed to inhibit therelease of the colorant (low values of the absorption), the moresensitive the detection for rivaroxaban becomes. The influencing factorspresent in plasma are not present in serum and urine.

DETAILED DESCRIPTION

According to the present invention, the term “factor Xa” does notunderlie a specific restriction and may include any activated factor Xaobtained from a natural source or via recombinant DNA technology, or abiologically active derivative thereof.

As used herein, the term “biologically active derivative” includes anyderivative of a protein, protein complex or polypeptide havingsubstantially the same functional and/or biological properties of FXasuch as binding properties, and/or the same structural basis, such as apeptidic backbone. The polypeptide sequences of the functionally activederivatives may contain deletions, additions and/or substitution ofamino acids whose absence, presence and/or substitution, respectively,do not have any substantial negative impact on the activity of thepolypeptide, e.g. amino acids which are located in a part of thepolypeptide sequence that does not contribute to the biological activityof the protein. Minor deletions, additions and/or substitutions of aminoacids of the respective polypeptide sequences which are not altering thebiological activity of said polypeptide are also included in the presentapplication as biologically active derivatives.

A factor Xa obtained from a natural source may be any factor Xa isolatedfrom a blood product derived from a mammal. In a preferred embodiment ofthe present application, the mammal is selected from the groupconsisting of mouse, human, rat, cat, dog, and monkey. In a particularlypreferred embodiment, the factor Xa is isolated from a blood product ofa human. In a preferred embodiment of the present application, thefactor Xa is isolated from a blood product selected from the groupconsisting of whole blood, serum, or plasma, including isolated bloodcompounds and processed blood products. A factor Xa obtained from anatural source may be a factor Xa obtained by isolating factor X from ablood product as defined above and subsequently activating the isolatedfactor X, to become factor Xa, e.g. by using any thromboplastin or byusing viper venom, such as Russell's viper venom.

The factor Xa according to the present invention may be produced by anymethod known in the art. This may include any method known in the artfor the production of recombinant DNA by genetic engineering, e.g. viareverse transcription of RNA and/or amplification of DNA. This includesmethods which comprise the recombinant production of factor X and thesubsequent activation of factor X, e.g. by using thromboplastin of byusing Russell's viper venom, in order to obtain factor Xa.

For example, the recombinant DNA coding for factor X, e.g. a plasmid,may also contain a DNA sequence encoding a selectable marker forselecting the cells which have been successfully transfected with theplasmid. In an example of the present invention, the plasmid may alsoconfer resistance to a selectable marker, e.g. to the antibiotic drugG418, by delivering a resistance gene, e.g. the neo resistance geneconferring resistance to G418.

The production of factor Xa may include any method known in the art forthe introduction of recombinant DNA into eukaryotic cells bytransfection, e.g. via electroporation or microinjection. For example,the recombinant expression of human factor X can be achieved byintroducing an expression plasmid containing the human factor X encodingDNA sequence under the control of one or more regulating sequences suchas a strong promoter, into a suitable host cell line by an appropriatetransfection method resulting in cells having the introduced sequencesstably integrated into the genome. The calcium-phosphateco-precipitation method is an example of a transfection method which maybe used according to the present invention.

The production of factor Xa may also include any method known in the artfor the cultivation of said transformed cells, e.g. in a continuous orbatchwise manner, and the expression of the factor X, e.g. constitutiveor upon induction. In one specific example of the present invention thenucleic acid coding for factor X contained in the host organism of thepresent invention is expressed via an expression mode selected from thegroup consisting of induced, transient, and permanent expression. Anyexpression system known in the art or commercially available can beemployed for the expression of a recombinant nucleic acid encodingfactor X, including the use of regulatory systems such as suitable, e.g.controllable, promoters, enhancers etc.

The production of factor Xa may also include any method known in the artfor the isolation of the protein, e.g. from the culture medium or byharvesting the transformed cells. For example, the factor X-producingcells can be identified by isolating single-cell derived populationsi.e. cell clones, via dilution after transfection and optionally viaaddition of a selective drug to the medium. After isolation theidentified cell clones may be cultivated until confluency in order toenable the measurement of the factor X content of the cell culturesupernatant by enzyme-linked immuno-sorbent assay (ELISA) technique.

Additionally, the production of factor Xa may include any method knownin the art for the purification of factor X or factor Xa, e.g. via anionexchange chromatography or affinity chromatography. In one preferredembodiment factor X can be purified from cell culture supernatants bysemi-affinity calcium-dependent anion exchange chromatography, e.g. inan endotoxin-free system. The purified factor Xa or factor X may beanalyzed by methods known in the art for analyzing recombinant proteins,e.g. the ELISA technique. In addition, the protein integrity andactivity may be assessed. It is within the knowledge of a person skilledin the art to choose the optimal parameters, such as buffer system,temperature and pH for the respective detection system to be used.

In one specific example of the present invention, the factor X accordingto the present invention is expressed in a host cell type with theability to perform posttranslational modifications. The ability toperform posttranslational modifications of factor X expressing host celllines may be for example analyzed by mass-spectrometric analysis.

The host cell type used for the recombinant production of factor Xa maybe any mammalian cell, preferably with the ability to performposttranslational modifications of factor X. There is no particularlimitation to the media, reagents and conditions used for culturing thecells in the cell culture used for the recombinant production of factorXa including culturing the cells in a continuous or batchwise manner.The desired factor X protein which has been expressed by the cells ofthe and which, dependent on the transfection/vector-system used, iscontained in the cells or secreted into the medium for culturing cells,can be isolated/recovered from the cell culture using methods known inthe art, as mentioned herein before.

The term “factor Xa” as used herein comprises any factor Xa which isobtained by producing and isolating factor X according to any methodavailable in the prior art and disclosed herein followed by a subsequentactivation of factor X, e.g. by using thromboplastin or Russell's vipervenom.

The term “at least one direct factor Xa inhibitor” as used hereinrelates to any naturally occurring or artificially synthesized inhibitorof factor Xa activity. In a preferred embodiment of the presentinvention, the factor Xa inhibitor is selected from the group,consisting at present of apixaban, betrixaban, edoxaban, otamixaban, andrivaroxaban and/or others in development. In a more preferred embodimentof the present invention, the factor Xa inhibitor is rivaroxaban.

The sample containing at least one direct factor Xa inhibitor providedin step (a) of the method for detecting at least one direct factor Xainhibitor in a sample according to the present invention, may be anysample which contains at least one direct factor Xa inhibitor, and whichdoes not contain citrated blood plasma. In one embodiment of the presentinvention, the sample may be derived from a naturally occurring system,preferably a sample containing a body fluid or components derived from abody fluid. In one embodiment of the present invention, the sample maybe a naturally occurring system such as a solution selected from thegroup consisting of serum, saliva, urine, bile, lymph, tissue, like e.g.bladder or kidney, cerebrospinal fluid and/or other body fluids. In apreferred embodiment of the present invention, the sample comprisesurine.

In a particularly preferred embodiment of the present invention, thesample comprises urine and the at least one direct factor Xa inhibitoris rivaroxaban.

Further, the sample may comprise a solution derived from naturallyoccurring systems, e.g. a solution containing isolated body fluidcompounds or processed body fluids. In another embodiment of the presentinvention, the sample may comprise cells or tissue samples obtained froma mammal. Methods for obtaining the above samples are known in the priorart.

The sample may be derived from a mammal, preferably a mammal selectedfrom the group consisting of human, mouse, rat, pig, cat, dog, horse,goat, cattle, cow, and monkey and/or others. In a preferred embodimentof the present invention, the sample is derived from a human. In anotherembodiment of the present invention, the sample contains isolated bodyfluid compounds or processed body fluids derived from a mammal,preferably a mammal selected from the group consisting of human, mouse,rat, pig, cat, dog, horse, goat, cattle, cow, and monkey and/or others.In a preferred embodiment of the present invention, the sample containsisolated body fluid compounds or processed body fluids derived from ahuman.

In a more preferred embodiment of the present invention, the sample isderived from a patient to which the factor Xa has been administeredbefore step (a) of the method for detecting at least one direct factorXa inhibitor in a sample according to the present invention. The patientcan be selected from the group consisting human, mouse, rat, pig, cat,dog, horse, goat, cattle, cow, and monkey and/or others. Mostpreferably, the patient is a human being. In a preferred embodiment ofthe present invention, the sample is a sample as defined herein.

In a preferred embodiment, the sample is pre-purified before step (a) ofthe method for detecting at least one direct factor Xa inhibitor in asample according to the present invention. In a more preferredembodiment of the present invention, the pre-purification comprises thestep of removing impurities that prevent the factor Xa inhibitor frombinding to factor Xa.

The composition containing at least one factor Xa provided in step (b)of the method for detecting at least one direct factor Xa inhibitor in asample according to the present invention may be any compositioncontaining at least one factor Xa and may also contain suitable buffersalts. In a preferred embodiment of the present invention thecomposition containing at least one factor Xa is isotonic within thephysiological limits of the pH value and may be of normal or low ionicstrength.

According to the present invention the chromogenic substrate conjugatedto a detectable substance provided in step (c) of the method fordetecting at least one direct factor Xa inhibitor in a sample accordingto the present invention is covalently linked to at least one detectablesubstance. The term “detectable substance” does not exhibit anyparticular limitation and may be selected from the group consisting ofradioactive labels, fluorescent dyes, compounds having an enzymaticactivity, magnetic labels, antigens, and compounds having a high bindingaffinity for a detectable substance. A compound having an enzymaticreactivity such as the enzyme luciferase which produces a light signalupon contact with the respective substrate can also be used as adetectable substance which may be linked covalently to said substrate.Coupling a detectable substance to an antigen allows the detection ofthe substance by an antibody/enzyme-complex (the enzyme being e.g.phosphatase) catalysing a detectable color reaction when using asuitable substrate. A compound with a high binding affinity for adifferent detectable substance such as biotin which binds to adetectable substance covalently linked to e.g. streptavidin, is afurther possibility for making a substance detectable. In a preferredembodiment of the present application, the detectable substance ispara-nitroaniline.

The chromogenic substrate conjugated to a detectable substance providedin step (c) of the method for detecting at least one direct factor Xainhibitor in a sample according to the present invention is anychromogenic substrate conjugated to a detectable substance which can becleaved by factor Xa so that the detectable substance is released fromthe chromogenic substrate. In a preferred embodiment of the presentinvention, the conjugation of the chromogenic substrate to thedetectable substance is via the linkerisoleucine-glutamine-glycine-arginine-X (Ile-Glu-Gly-Arg-X) or via thelinker isoleucine-aspartic acid-glycine-arginine-X (Ile-Asp-Gly-Arg-X),wherein X is any amino acid except of proline. In a preferred embodimentof the present application, the chromogenic substrate conjugated to adetectable substance is an amino acid sequence which contains thesequence Ile-Glu-Gly-Arg-X or Ile-Asp-Gly-Arg-X, wherein X is any aminoacid except of proline, at the site where the detectable substancebinds, provided that the structure of the chromogenic substrateconjugated to a detectable substance is such that factor Xa cleaves thesequence Ile-Glu-Gly-Arg-X or Ile-Asp-Gly-Arg-X under physiologicalconditions at room temperature.

In a preferred embodiment of the present invention, the chromogenicsubstrate conjugated to a detectable substance is a fluorogenicsubstance. In a more preferred embodiment of the present invention, thefluorogenic substance is selected from the group consisting ofN-Benzoyl-Ile-Glu-Gly-Arg p-nitroanilide acetate salt,N-benzoyl-L-isoleucyl-L-glutamyl-L-glycyl-L-arginine-para-nitroanilinehydrochloride, and Boc-Ile-Glu-Gly-Arg-7-amido-4-methylcoumarinhydrochloride. In a particularly preferred embodiment of the presentinvention the chromogenic substrate conjugated to a detectable substanceisN-benzoyl-L-isoleucyl-L-glutamyl-L-glycyl-L-arginine-para-nitroanilinehydrochloride.

The conditions suitable for the binding of the at least one directfactor Xa inhibitor to factor Xa and which allow the factor Xa torelease the detectable substance from the chromogenic substrate in step(d) of the method for detecting at least one direct factor Xa inhibitorin a sample according to the present invention may take place in abuffer solution. If a buffer solution is used in step (d) of the methodfor detecting at least one direct factor Xa inhibitor in a sampleaccording to the present invention, it may contain any compound whichdoes not negatively affect inhibitor-factor Xa-complex forming and therelease the detectable substance from the chromogenic substrate byfactor Xa. In a preferred embodiment of the method for detecting atleast one direct factor Xa inhibitor in a sample according to thepresent invention, the conditions in step (d) comprise the use of a thebuffer solution which is isotonic and within the physiological limits ofthe pH value. It may be of normal or low ionic strength. The buffersalts used in step (d) of the method for detecting at least one directfactor Xa inhibitor in a sample according to the present invention maybe any buffer salt as long as said buffer salt does not negativelyaffect the inhibitor-factor Xa-complex forming and the release thedetectable substance from the chromogenic substrate by factor Xa.

Step (d) of the method for detecting at least one direct factor Xainhibitor in a sample according to the present invention may be carriedout under any conditions suitable for binding of the at least one directfactor Xa inhibitor to factor Xa and which allow the factor Xa torelease the detectable substance from the chromogenic substrate withoutany limitation. This comprises e.g. any suitable temperature, timeperiod and agitation of the buffer solution. In a preferred embodimentof the present invention, the incubation is carried out at a temperatureranging from about 20° C. to about 37° C. for from about 1 to about 30minutes. In a more preferred embodiment of the present invention, theincubation is carried out at about 37° C. for about 20 minutes.

In a preferred embodiment, the method for detecting at least one directfactor Xa inhibitor in a sample according to the present inventionincludes the step of removing the chromogenic substrate after step (d)and before step (e). The chromogenic substrate can be removed by methodswell known in the art. Examples for the removal of the chromogenicsubstrate are, but not limited to, for example the use of antibodies orenzymes specifically binding the chromogenic substrate. In a preferredembodiment of the present application, the antibodies or enzymes,preferably coagulation enzymes, specifically binding the chromogenicsubstrate are bound to a support as defined herein. Further, thechromogenic substrate may be covalently linked to a compound with a highbinding affinity for a different compound such as biotin which binds toa compound covalently linked to e.g. streptavidin, or to a magneticcompound is a further possibility for removing the chromogenicsubstrate.

The removal of the chromogenic substrate can be carried out by standardmethods. For example, if the one or more or all of the antibodies orenzymes, preferably coagulation enzymes, specifically binding thechromogenic substrate is conjugated to biotin, the chromogenic substratecan be removed by binding the biotin to streptavidin and the subsequentremoval of the biotin-streptavidin-complex, e.g. by centrifugation or,if the streptavidin is conjugated to a suitable support, like a resinmaterial, by column chomatography. As an alternative, if the antibodiesor enzymes, preferably coagulation enzymes, specifically binding thechromogenic substrate are covalently linked to a magnetic compound, thechromogenic substrate can be removed by binding said chromogenicsubstrate via a magnetic compound having the opposite polarity. Thereaction conditions to perform the removal of the chromogenic substratedepend upon the removal method selected. It is within the knowledge ofthe person skilled in the art to choose the optimal parameters, such asbuffer system, temperature and pH for the respective removal system tobe used.

The reaction conditions for measuring the amount of released detectablesubstance in step (e) of the method for detecting at least one directfactor Xa inhibitor in a sample according to the present inventiondepend upon the detection method selected. It is within the knowledge ofthe person skilled in the art to choose the optimal parameters, such asbuffer system, temperature and pH for the respective detection system tobe used.

The measuring step (e) of the above-defined method may comprise one ormore detection method(s) selected from the group consisting ofimmunoblotting, immunoprecipitation, immunocapture, monoclonal antibodyimmobilization of platelet antigens or enzyme linked immuno sorbentassay (ELISA), flow cytometry, protein array technology, spectroscopy,mass spectrometry, chromatography, surface plasmonic resonance,fluorescence extinction and/or fluorescence energy transfer. Thedetection method for measuring the detectable substance can, forexample, be selected from the group consisting of an enzyme assay, achromogenic assay, a lumino assay, a fluorogenic assay, and aradioimmune assay. The reaction conditions to perform detection of thedetectable label depend upon the detection method selected. It is withinthe knowledge of the person skilled in the art to choose the optimalparameters, such as buffer system, temperature and pH for the respectivedetection system to be used. In a preferred embodiment of the presentapplication, the detectable substance para-nitroaniline and the releasedpara-nitroaniline is detected via measuring the absorption of the sampleat 405 nm.

If the detectable substance is detected via antibodies specificallybinding the detectable substance, the antibodies may be immobilized on asupport, preferably a solid support. The term “support” does not haveany specific limitations, and relates, for example, to an insolublepolymer material, which can be an organic polymer, such as polyamide ora vinyl polymer (e.g. poly(meth)acrylate, polystyrene and polyvinylalcohol, or derivatives thereof), a natural polymer such as cellulose,dextrane, agarose, chitin and polyamino acids, or an inorganic polymer,such as glass or metallohydroxide. The support can be in the form of amicrocarrier, particles, membranes, strips, paper, film, pearls orplates, such as microtiter plates or microarrays. The term “microarray”as used herein may mean any arrangement of the antibodies in addressablelocations on a support resulting in a so-called “biochip”. The supportmay also be used as resin material, which can be used in a columnchromatography.

In a preferred embodiment of the present invention, the method fordetecting at least one direct factor Xa inhibitor in a sample accordingto the present invention is a method for point-of-care testing using asample which is not plasma or whole blood. When the method for detectingat least one direct factor Xa inhibitor in a sample according to thepresent invention is a method for point-of-care testing, the compositioncontaining factor Xa provided in step (b) of the method for detecting atleast one direct factor Xa inhibitor in a sample according to thepresent invention and/or the composition containing a chromogenicsubstrate conjugated to a detectable substance provided in step (c) ofthe method for detecting at least one direct factor Xa inhibitor in asample according to the present invention are immobilized on a teststrip. The mixing of the sample of step (a) with the composition of step(b) and/or the composition of step (c) in step (d) is obtained byapplying a test sample as defined herein on the respective position onthe test strip, on which the composition containing factor Xa and/or thecomposition containing composition containing a chromogenic substrateconjugated to a detectable substance is immobilized. The measuring ofthe amount of released substance according to step (e) of the method fordetecting at least one direct factor Xa inhibitor in a sample accordingto the present invention is accomplished by inserting the test stripinto an instrument being suitable for measuring of the amount ofreleased substance. In a preferred embodiment of the present invention,the instrument is a transportable, portable or handheld instrument.

In a preferred embodiment of the present invention, the method fordetecting at least one direct factor Xa inhibitor in a sample accordingto the present invention is a method for point-of-care testing and thecomposition containing factor Xa provided in step (b) of the method fordetecting at least one direct factor Xa inhibitor in a sample accordingto the present invention is immobilized on a test strip and thecomposition containing a chromogenic substrate conjugated to adetectable substance provided in step (c) of the method for detecting atleast one direct factor Xa inhibitor in a sample according to thepresent invention is not immobilized on the test strip. The mixing ofthe sample of step (a) with the composition of step (b) in step (d) isobtained by applying a test sample as defined herein on the respectiveposition on the test strip, on which the composition containing factorXa is immobilized.

In one embodiment, the test strip is inserted into an instrument beingsuitable for providing the composition of step (c) and for measuring ofthe amount of released substance. The mixing of the sample of step (a)and the composition of step (b) with the composition of step (c) in step(d) is obtained by applying the composition of step (c) on therespective position on the test strip, on which the compositioncontaining factor Xa is immobilized and already mixed with the testsample, in the test instrument. The measuring of the amount of releasedsubstance according to step (e) of the method for detecting at least onedirect factor Xa inhibitor in a sample according to the presentinvention is accomplished by the instrument into which the test striphas been inserted. In a preferred embodiment of the present invention,the instrument is a transportable, portable or handheld instrument.

In another embodiment, the composition of step (c) is applied manually,for example by using a pipette. The mixing of the sample of step (a) andthe composition of step (b) with the composition of step (c) in step (d)is obtained by applying the composition of step (c) on the respectiveposition on the test strip, on which the composition containing factorXa is immobilized and already mixed with the test sample. The measuringof the amount of released substance according to step (e) of the methodfor detecting at least one direct factor Xa inhibitor in a sampleaccording to the present invention is accomplished by assessing opticalchanges of the mixed sample, for example a change of color. Thisassessment can be carried out, for example by comparing the opticalappearance of the mixture with the optical appearance of a negativecontrol and/or a positive control, which can be for example provided onthe test strip or in a manual provided by the manufacturer of the teststrip. The optical changes of the mixed sample can also be carried out,for example by comparing the optical appearance of the mixture with theoptical appearance of positive controls having different concentrationsof the direct factor Xa inhibitor, for example obtained using a standarddilution series.

In another preferred embodiment of the present invention, the method fordetecting at least one direct factor Xa inhibitor in a sample accordingto the present invention is a method for point-of-care testing and thecomposition containing a chromogenic substrate conjugated to adetectable substance provided in step (c) of the method for detecting atleast one direct factor Xa inhibitor in a sample according to thepresent invention is immobilized on a test strip and the compositioncontaining factor Xa provided in step (b) of the method for detecting atleast one direct factor Xa inhibitor in a sample according to thepresent invention is not immobilized on the test strip. The mixing ofthe sample of step (a) with the composition of step (c) in step (d) isobtained by applying a test sample as defined herein on the respectiveposition on the test strip, on which the composition containing achromogenic substrate conjugated to a detectable substance isimmobilized.

In one embodiment, the test strip is inserted into an instrument beingsuitable for providing the composition of step (b) and for measuring ofthe amount of released substance. The mixing of the sample of step (a)and the composition of step (c) with the composition of step (b) in step(d) is obtained by applying the composition of step (b) on therespective position on the test strip, on which the compositioncontaining a chromogenic substrate conjugated to a detectable substanceis immobilized and already mixed with the test sample, in the testinstrument. The measuring of the amount of released substance accordingto step (e) of the method for detecting at least one direct factor Xainhibitor in a sample according to the present invention is accomplishedby the instrument into which the test strip has been inserted. In apreferred embodiment of the present invention, the instrument is atransportable, portable or handheld instrument.

In another embodiment, the composition of step (b) is applied manually,for example by using a pipette. The mixing of the sample of step (a) andthe composition of step (b) with the composition of step (c) in step (d)is obtained by applying the composition of step (b) on the respectiveposition on the test strip, on which the composition containing achromogenic substrate conjugated to a detectable substance isimmobilized and already mixed with the test sample. The measuring of theamount of released substance according to step (e) of the method fordetecting at least one direct factor Xa inhibitor in a sample accordingto the present invention is accomplished by assessing optical changes ofthe mixed sample, for example a change of color. This assessment can becarried out, for example by comparing the optical appearance of themixture with the optical appearance of a negative control and/or apositive control, which can be for example provided on the test strip orin a manual provided by the manufacturer of the test strip. The opticalchanges of the mixed sample can also be carried out, for example bycomparing the optical appearance of the mixture with the opticalappearance of positive controls having different concentrations of thedirect factor Xa inhibitor, for example obtained using a standarddilution series.

When the method for detecting at least one direct factor Xa inhibitor ina sample according to the present invention is a method forpoint-of-care testing, the direct factor Xa inhibitor, the sample, thecomposition containing factor Xa, the factor Xa, the compositioncontaining a chromogenic substrate conjugated to a detectable substance,the chromogenic substance, the detectable substance, and/or each ofsteps (a) to (e) are preferably as defined herein. In a particularlypreferred embodiment of the present invention, the chromogenic substrateconjugated to a detectable substance provided in step (c) is selectedfrom the group consisting of radioactive labels, compounds having anenzymatic activity, magnetic labels, antigens, and compounds having ahigh binding affinity for a detectable substance.

In a preferred embodiment of the present invention, the method fordetecting at least one direct factor Xa inhibitor in a sample accordingto the present invention is a method for point-of-care testing asdefined herein, the sample is urine and the direct factor Xa inhibitoris rivaroxaban. Preferably, (i) the composition containing factor Xaprovided in step (b) or (ii) the composition containing a chromogenicsubstrate conjugated to a detectable substance provided in step (c) areimmobilized on a test strip. In a preferred embodiment, the chromogenicsubstrate conjugated to a detectable substance isN-benzoyl-L-isoleucyl-L-glutamyl-L-glycyl-L-arginine-para-nitroanilinehydrochloride. The test strip is inserted into the urine sample. Then,(i) in case the composition containing factor Xa provided in step (b) isalready immobilized on the test strip, the composition containing achromogenic substrate conjugated to a detectable substance provided instep (c) is added, or (ii) in case the composition containing achromogenic substrate conjugated to a detectable substance provided instep (c) is already immobilized on the test strip, the compositioncontaining factor Xa provided in step (b) is added. The more opticalchanges of the mixed sample can be observed, preferably the more yellowthe mixed sample turns, the less factor Xa inhibitors are in the sample.

The antibodies specifically binding the detectable substance or theantibodies specifically binding the chromogenic substrate, if thechromogenic substrate is removed, can be immobilized on the supportdirectly by covalent coupling or via a carrier such as a linker moleculeor an antibody immobilized on the support. Further, the antibodiesspecifically binding the detectable substance or the support may becovalently linked to a detectable label which may be any suitabledetectable label known in the art. In a preferred embodiment of thepresent invention, the detectable label is biotin or a magneticsubstance.

In a preferred embodiment of the present invention, the method fordetecting at least one direct factor Xa inhibitor in a sample accordingto the present invention further contains after step (e) a step

(f) determining the amount of factor Xa inhibitor in the sample bycorrelating the amount of released detectable substance with the amountof factor Xa inhibitor in the sample.

The amount of released detectable substance decreases with an increaseof factor Xa inhibitor in the sample.

The quantification of the detectable substance, preferably resulting inthe determination of the amount of factor Xa inhibitor in the sample,can be carried out by standard methods. In a preferred embodiment of thepresent invention, the amount of factor Xa inhibitor in the sample iscalculated from a calibration curve obtained by a factor Xa inhibitor indefined amounts.

The present invention further relates to a use of a compositioncontaining factor Xa for monitoring the course of treatment with atleast one direct factor Xa inhibitor in a patient. In another preferredembodiment the use of a composition containing factor Xa for monitoringthe course of treatment with at least one direct factor Xa inhibitor ina patient comprises the method for detecting at least one direct factorXa inhibitor in a sample according to the present invention as definedherein. In a preferred embodiment of the present invention, the factorXa, the factor Xa inhibitor, and/or the patient is as defined herein. Ina preferred embodiment of the present invention, the course of treatmentis monitored by detecting at least one direct factor Xa inhibitor in asample, more preferably in a test sample comprising serum, urine, or anyother body fluid.

The present invention further relates to a composition containing factorXa for use in monitoring the course of treatment with direct factor Xainhibitors in a patient for diagnostic purposes. In a preferredembodiment, the present invention relates to a composition containingfactor Xa for use in monitoring the course of treatment with directfactor Xa inhibitors in a patient for diagnostic purposes, wherein anelevated risk for thrombosis is associated with an increase of factor Xainhibitors in the sample. In a preferred embodiment of the presentinvention, the factor Xa, the factor Xa inhibitor, and/or the patient isas defined herein.

A further embodiment of the present invention relates to a diagnostickit for monitoring the course of treatment with direct factor Xainhibitors in a patient comprising a composition containing factor Xaand composition containing a chromogenic substrate conjugated to adetectable substance. In a preferred embodiment of the presentinvention, the kit contains further any means for carrying out themethod for detecting at least one direct factor Xa inhibitor in a sampleaccording to the present invention as defined herein. In particular, thekit may contain one or more of the following: a chromogenic substrateconjugated to a detectable substance as defined herein, antibodiesspecifically binding the detectable substance as defined herein, factorXa and modified factor Xa proteins specifically binding the chromogenicsubstrate as defined herein, a support with immobilized antibodies orfactor Xa enzymes specifically binding the detectable substance asdefined herein, a support with immobilized antibodies specificallybinding the chromogenic substrate as defined herein, buffer solutions asdefined herein, reaction containers, and/or means for measuring theamount of released detectable substance as defined herein, includingbuffers, when appropriate. In a preferred embodiment of the presentinvention, the factor Xa, the direct factor Xa inhibitor, and/or thepatient is as defined herein.

It one of the aspects underlying the present invention to measure theconcentration/activity of a direct factor Xa inhibitor like rivaroxabanwith a chromogenic test from samples for measuring other bloodcomponents (liver parameters, kidney parameters, cholesterol, etc.) orblood count without an additional blood withdrawal for the bloodcoagulation. Using the present invention the concentration of a factorXa inhibitor like rivaroxaban in urine without a further bloodwithdrawal can be determined. Instead of rivaroxaban, other directinhibitors of factor Xa in matrices other than plasma can be measuredwith a chromogenic test.

Direct factor Xa inhibitors inhibit exogenous factor Xa also without thepresence of antithrombin or factor X or endogenous factor Xa. Thus, themeasurement from serum, urine, and other matrices becomes possible. Theinvention solves the problem that the direct factor Xa inhibitorsinhibit the colorant para-nitroaniline from a chromogenic substrate in adose-dependent manner only in the presence of exogenous factor Xa. Aseparate blood withdrawal for the coagulation measurement is notnecessary any more using the method according to the present invention.For an examination in urine, a blood withdrawal can be omitted. The riskof side effects of a blood withdrawal is reduced (serum tube, bloodcount tube) or eliminated (urine).

It is essential for the present invention that rivaroxaban and otherdirect factor Xa inhibitors inhibit exogenously added factor Xa withoutthe presence of other coagulation proteins. Thereby, theconcentration/activity of factor Xa inhibitors, like rivaroxaban, inserum and other biological matrices can be detected.

The advantages of the invention are that using the method according tothe present invention, the side effects and risks associated with ablood withdrawal are reduced (detection in serum) or eliminated(detection in urine), and the special taking of a “coagulation tube” isnot necessary.

The basis for the present invention is the fact that for a photometricdetermination of the inhibition of factor Xa no fibrinogen, and for thedirect inhibitors of factor Xa no antithrombin are necessary ascofactors. It is essential for the present invention that theconcentration/activity of medicaments that directly inhibit factor Xacan be quantified via the inhibition of exogenous factor Xa in and onmedia/matrices by means of a factor Xa specific chromogenic substrate,which are not plasma (FIG. 1).

The present invention will now be further illustrated in the followingexamples without being limited thereto.

EXAMPLES Example 1

a) Measurement of Rivaroxaban in Serum with Factor Xa and S2222Reagents: Solution 1: Aqua destillata

-   -   Solution 2:    -   Tris Buffer Tris 6.06 g    -   NaCl 10.23 g    -   EDTA 2.79 g    -   pH 8.4    -   ad 1000 ml Aqua destillata    -   Solution 3:    -   serial dilution Rivaroxaban with 0 ng/ml, 50 ng/ml, 100 ng/ml,        150 ng/ml, 300 ng/ml, 500 ng/ml, 700 ng/ml, dissolved in serum    -   Solution 4:    -   FXa 71 nkat (Chromogenix, Essen Germany)    -   dissolved in 10 ml A. dest.    -   Solution 5:    -   S-2222 25 mg (Chromogenix, Essen Germany)    -   dissolved in 33.7 ml A. dest    -   Solution 6:    -   acidic acid: 50%        Test description: standard curve    -   25 μl serum with known concentration of Rivaroxaban,    -   1:15 diluted in tris buffer

+25 μl FXa

-   -   2 min. incubation at 37° C.

+50 μl S-2222

-   -   20 min. incubation at 37° C.        +25 μl Essigsäure 50%

Measurement at 405 nm

Preparation of a standard curve (OD versus ng/ml)Test description: determination of serum samples

-   -   25 μl serum, containing Rivaroxaban, 1:15 diluted in tris buffer

+25 μl FXa

-   -   2 min. incubation at 37° C.

+50 μl S-2222

-   -   20 min. incubation at 37° C.        +25 μl acidic acid 50%

Measurement at 405 nm

Calculation of the concentration of Rivaroxaban was carried out usingthe standard curve. The concentration of Rivaroxaban was determined bythe optical density (OD) of the sample.

b) Measurement of Rivaroxaban in Urine with Factor Xa and S2222

Reagents are the same as in the measurement of Rivaroxaban in serum.

Test description: standard curve

-   -   25 μl Rivaroxaban in urine

+25 μl FXa

-   -   2 min. incubation at 37° C.

+50 μl S-2222

-   -   20 min. incubation at 37° C.        +25 μl acidic acid 50%

Measurement at 405 nm

Preparation of a standard curve (OD versus ng/ml)Test description: determination of urine samples

-   -   25 μl urine

+25 μl FXa

-   -   2 min. incubation at 37° C.

+50 μl S-2222

-   -   20 min. incubation at 37° C.        +25 μl acidic acid 50%

Measurement at 405 nm

Calculation of the concentration of Rivaroxaban was carried out usingthe standard curve. The concentration of Rivaroxaban was determined bythe optical density (OD) of the sample.

The direct factor Xa inhibitor rivaroxaban was added to samples ofplasma, serum, and urine in different concentrations. The reagentsfactor Xa (from Chromogenix and Coachrom) and chromogenic substrate(S2222 from Chromogenix and CS1156 from Coachrom) were added to thesamples, incubated and measured in a photometer at a wavelength of 405nanometers (nm). Rivaroxaban inhibits the activity of factor Xa and thusthe release of para-nitroaniline from the chromogenic substrate S2222 ina dose-dependent manner (FIG. 2).

Due to missing antithrombin and other cofactors for heparins and missingfactor X, factor Xa and other coagulation proteins in the assay run, thedetection of rivaroxaban and other factor Xa inhibitors is lesssensitive and susceptible to influences (FIG. 2).

Our studies have shown that rivaroxaban in serum and urine can bemeasured with a chromogenic test without the addition of plasma.

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 15. Diagnostic kit for monitoring the course of treatment with factor Xa inhibitors in a patient comprising a composition containing factor Xa and composition containing a chromogenic substrate conjugated to a detectable substance.
 16. The diagnostic kit according to claim 15, wherein the course of treatment with factor Xa inhibitors in a patient is measured in a sample that does not contain citrated blood plasma.
 17. The diagnostic kit according to claim 15, wherein the course of treatment with factor Xa inhibitors in a patient is measured in a sample that contains a body fluid.
 18. The diagnostic kit according to claim 16, wherein the body fluid is selected from the group consisting of serum and urine.
 19. The diagnostic kit according to claim 15, wherein the chromogenic substrate conjugated to a detectable substance is a chromogenic substrate having para-nitroaniline conjugated via the linker Ile-Glu-Gly-Arg-X or via the linker Ile-Asp-Gly-Arg-X, wherein X is any amino acid except of proline
 20. The diagnostic kit according to claim 15, wherein the chromogenic substrate conjugated to a detectable substance is selected from the group consisting of N-Benzoyl-Ile-Glu-Gly-Arg p-nitroanilide acetate salt, N-benzoyl-L-isoleucyl-L-glutamyl-L-glycyl-L-arginine-para-nitroaniline hydrochloride, Boc-Ile-Glu-Gly-Arg-7-amido-4-methylcoumarin hydrochloride, 4-Nitrophenyl 4-guanidinobenzoate hydrochloride.
 21. The diagnostic kit according to claim 15, wherein the chromogenic substrate conjugated to a detectable substance is N-Benzoyl-Ile-Glu-Gly-Arg p-nitroanilide acetate salt.
 22. The diagnostic kit according to claim 15, wherein the factor Xa inhibitor is selected from the group consisting of apixaban, edoxaban, otamixaban, and rivaroxaban.
 23. The diagnostic kit according to claim 15, wherein the factor Xa inhibitor is rivaroxaban.
 24. The diagnostic kit according to claim 15, which is a point-of-care testing.
 25. The diagnostic kit according to claim 24, wherein the composition containing factor Xa and/or the composition containing a chromogenic substrate conjugated to a detectable substance are immobilized on a test strip.
 26. The diagnostic kit according to claim 15, wherein an elevated risk for thrombosis is associated with an increase of factor Xa inhibitors in the sample. 